Liquid chromatography (LC) is a well-established analytical technique for separating components of a fluidic mixture for subsequent analysis and/or identification, in which a column is packed with a stationary phase material that typically is a finely divided solid or gel such as small particles with diameters of a few microns. A variety of different detectors can be used to detect the analytes separated on an LC column. Additionally, the separated components may be passed from the liquid chromatography column into other types of analytical instruments for further analysis, e.g., liquid chromatography-mass spectrometry (LC-MS) separates compounds chromatographically before they are introduced to an ion source of a mass spectrometer.
Mass spectrometry (“MS”) is an analytical technique used to measure the mass-to-charge ratio of gas phase ions. This is achieved by ionizing a sample and separating ions of differing masses and recording their relative abundance by measuring intensities of ion flux. Electrospray ionization (ESI) is a commonly applied ionization technique when dealing with biomolecules such as peptides and proteins. The electrospray process creates highly-charged droplets that, under evaporation, create ions representative of the species contained in a sample solution.
Mass spectrometry-based proteomics, particularly liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS), has recently become the technique of choice for rapid identification and characterization of proteins in biological systems. Moreover multiple approaches now exist to conduct these experiments in a semi-quantitative manner to monitor changes in protein expression and specific post-translational events as a function of biological perturbation. Despite its advantages discussed above, mass spectrometry suffers from limited dynamic range (e.g., the inability to detect peptides spanning an abundance ratio greater than 5000:1 in a single scan/spectrum) and finite acquisition rate (e.g., the inability to acquire MS/MS data at a rate sufficient to provide sequence information on all peptides in a complex mixture during a single LC-MS/MS run, regardless of their relative abundance). These limitations, combined with current trends towards analysis of increasingly complex mixtures (biomarkers, signaling pathways, etc) continue to push the limits of the ubiquitously used 75 μm and 100 μm I.D. (i.e., inner diameter) fused silica capillary columns packed with widely available 5 and 10 μm diameter reversed phase HPLC resins.